Once TMB is added the color starts to become blue, but what is the best time to add stop solution? Any method to monitor it or determine it? Why?
Thank you !
Many people just wait 15min or follow the instructions, if it is a kit, and are happy if the sample values are within the standard curve and there is no saturation.
The color development depends on temperature, so it can make sense to check / control the lab temp and to make sure that it does not pull through or to use a dedicated microplate incubator. Some microplate readers have this option.
Overall color development also depends on the quality of the HRP (-> RZ, Reinheitszahl) and the labeling ratio of the antibodies / conjugates used.
To optimize your assay, run a standard curve (or parts of it) and measure the plate every 2 minutes, to see how the color develops and when it goes into saturation for a given standard concentration. For optimal results, you can calculate some statistics to determine the incubation time for best signal-to-noise ratio, data fluctuation and detection range.
You can monitor the absorbance of TMB @ 605nm before addition of the stop solution.
It depends on the assay kit that you are using. They usually come with time specifications but if not, you can try leaving the plates for 5minuntes, incubating them at room temperature. Some plate detections are faster than other, so I think monitoring the plates during this incubation time will help!
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays